|Molecular Vision 2003;
Received 11 March 2003 | Accepted 24 April 2003 | Published 14 May 2003
Amyloid-β is found in drusen from some age-related macular degeneration retinas, but not in drusen from normal retinas
Tzvete Dentchev,1 Ann H. Milam,1 Virginia M.-Y.
Lee,2 John Q. Trojanowski,2
Joshua L. Dunaief1
1F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute and the 2Department of Pathology and Laboratory Medicine, Institute on Aging, Alzheimer's Disease Center, Center for Neurodegenerative Disease Research, University of Pennsylvania, Philadelphia, PA
Correspondence to: Joshua L. Dunaief, 305 Stellar-Chance Labs, 422 Curie Boulevard, Philadelphia, PA, 19104; Phone: (215) 898-5235; FAX: (215) 573-3918; email: firstname.lastname@example.org
Purpose: Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. Increased understanding of the pathogenesis is necessary. Amyloid-beta (Aβ), a major extracellular deposit in Alzheimer's disease plaques, has recently been found in drusen, the hallmark extracellular deposit in AMD. The goal of this study was to characterize the distribution and frequency of Aβ deposits in drusen from AMD and normal post mortem human retinas to gain additional insight about the potential role of Aβ in AMD pathogenesis.
Methods: Immunocytochemistry was performed with three Aβ antibodies on sections from 9 normal and 9 AMD (3 early, 3 geographic atrophy, 3 exudative AMD) retinas. Five sections from each eye were evaluated. Aβ positive deposits in drusen were identified using epifluorescence and confocal microscopy. Antibodies were pre-adsorbed with Aβ peptide to verify specificity. Some sections were stained with PAS-hematoxylin to aid in evaluation of morphology.
Results: To test and optimize immunocytochemistry, Aβ was detected in amyloid plaques from Alzheimer's brains. Aβ label was blocked by pre-adsorption of antibody with Aβ peptide, verifying specificity. Four of the 9 AMD retinas and none of the 9 normal retinas had Aβ positive drusen. Two of the early AMD eyes had a few Aβ positive drusen, each with a few Aβ-containing vesicles, and 2 of the geographic atrophy (GA) eyes had many Aβ positive drusen with many Aβ containing vesicles.
Conclusions: Aβ was present in 4 of 9 AMD eyes. Within these eyes, Aβ localized to a subset of drusen. None of the 9 normal eyes surveyed, some of which had small drusen, were A beta positive. Aβpositive vesicles were most numerous in GA eyes at the edges of atrophy, the region at risk for further degeneration. These results suggest that Aβ in drusen correlates with the location of degenerating photoreceptors and retinal pigment epithelium (RPE) cells. Further work will be necessary to determine whether Aβ deposition in drusen may contribute to or result from retinal degeneration.
Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly , yet its pathogenesis remains poorly understood. Current treatments are inadequate and would be improved by a better understanding of the molecular events causing the degeneration. It may be possible to gain insight into AMD pathogenesis by exploring similarities to another age-related disease of the central nervous system: Alzheimer's disease (AD). Extracellular amyloid beta (Aβ) deposition, oxidative stress, and inflammation are important molecular mediators of Alzheimer's disease . Similarly, recent evidence implicates Aβ, oxidative stress, and inflammatory processes in the pathogenesis of AMD. A role for Aβ in AMD has been suggested by the recent finding of Aβ in drusen, the extracellular deposits that are the earliest sign of AMD . This Aβ in drusen might increase oxidative stress and inflammation in AMD, as it does in AD plaques [2,3].
Cell loss in AMD can occur by apoptosis of retinal pigment epithelial cells followed by apoptosis of photoreceptors . The apoptotic stimulus for retinal pigment epithelium (RPE) death may involve drusen, located just beneath the RPE. It is possible that Aβ deposition in drusen is a cause or a consequence of RPE and photoreceptor degeneration. While solid evidence has been presented that vesicular Aβ deposits are present in drusen , we sought to learn more about the prevalence of Aβ deposits in drusen and the association of these deposits with AMD pathology. We report immunocytochemical analysis of Aβ in 9 normal and 9 AMD post mortem retinas.
Source of tissue and population profile
Most post mortem eyes were obtained through the Foundation Fighting Blindness (FFB) eye donor program. The eye pathology reports provided the patient age, gender, brief ocular and medical history, cause of death, and post mortem interval. A few eyes were obtained directly from eye banks. One donor had a potentially relevant co-morbid condition; patient 99-35 had insulin-dependent diabetes mellitus (but no Aβ-positive vesicles). The histopathologic studies followed the tenets of the Declaration of Helsinki, and informed consent was obtained from all eye donors ante mortem. Approval for research on human post mortem donor eyes was obtained from the University of Pennsylvania.
Brain samples from AD patients followed clinically by the National Institute on Aging-funded Penn Alzheimer's Disease Center Core (ADCC) were obtained following similar consent procedures and AD was confirmed by ADCC investigators according to established consensus criteria (National Institute on Aging and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer's Disease).
Tissue processing and histology
Upon enucleation, a small incision was made in the pars plana and the eyes were fixed in 4% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M phosphate buffer for several days. The fixed eyes were transferred to 2% paraformaldehyde for storage. A block of tissue containing the optic disc and macula approximately 1 cm in length and 0.5 cm in width was processed as serial 10 mm thick cryosections as previously described .
Immunocytochemistry and pre-adsorption
Immunocytochemistry (ICC) was performed using secondary antibody conjugated to Cy3 (red fluorescence emission) as described previously . Five sections from each retina spaced at approximately 400 μm intervals were labeled with monoclonal anti-Aβ antibodies 4G8 and 6E10 (Chemicon, Temecula, CA; 1:1000) and polyclonal antibody pAb 2332 that recognizes multiple species of human Aβ as described by Uryu et al. and additional references therein . Anti-rhodopsin mAb 4D2 (provided by R. Molday; 1:80) was also used. Peptide blocking experiments were performed with a five-fold molar excess of Aβ peptide 1-42 (BioSource International, Camarillo, CA), pre-adsorbing with the primary antibody at working dilution for 1 h with gentle agitation at room temperature. Images were obtained with a Nikon TE300 epifluorescence microcope equipped with a Spot RT Slider camera and ImagePro software. Images were also obtained with a Zeiss LSM 510 confocal microscope.
To learn more about the prevalence of Aβ deposits in drusen and the association of these deposits with AMD pathology we used ICC to detect Aβ in normal and AMD post mortem retinas. To test and optimize ICC, we first detected Aβ in amyloid plaques from Alzheimer's brains. Aβ label in plaques (Figure 1A, arrows) was blocked by pre-adsorption of antibody with Aβ peptide, verifying specificity (Figure 1B). The pink color remaining in Figure 1B is from tissue autofluorescence. Plaques were detected with our polyclonal antibody pAb 2332 as well as commercially available antibodies mAb 6E10 and 4G8 (not shown).
Sections from each of 18 retinas were then immunostained with Aβ antibodies. Nine of the retinas were from donors with normal eyes and 9 with AMD as defined by histologic detection of drusen and at least some RPE and/or photoreceptor loss. Three retinas had drusen and minimal RPE and photoreceptor loss (early AMD), 3 had geographic atrophy (GA), and 3 had exudative AMD (Table 1). Four of the 9 AMD retinas and none of the normal retinas had Aβ positive drusen. Two early AMD retinas had a few Aβ positive vesicles in a few drusen, and 2 GA retinas had many Aβ positive vesicles in many drusen. All retinas with drusen contained at least some Aβ-negative drusen, as determined by immunolabeling serial sections through entire drusen.
Retina 00-11, from a donor with GA throughout much of the macula, had multiple large drusen at the temporal edge of the macula, where photoreceptors and RPE cells were still present, although there was some loss of photoreceptor nuclei. Aβ positive vesicles were detected in these drusen with all three anti-Aβ antibodies, but not with negative control anti-rhodopsin (red label-Figure 2A,B,D). Vesicles were not detected by the secondary antibody alone (Figure 2E). Pre-adsorption of the anti-Aβ antibody with Aβ peptide greatly diminished vesicular labeling (Figure 2C,G).
Retina 99-30, also from a donor with GA throughout much of the macula, had many vesicles in a region of sub-RPE deposit beginning at the margin of atrophy, 1 mm temporal to the foveola and continuing another 2 mm temporal. The concentration of vesicles was highest near the margin of atrophy (Figure 3A). Pre-adsorption of antibody with Aβ peptide blocked vesicular labeling, demonstrating specificity (Figure 3B).
Two retinas with minimal photoreceptor and RPE atrophy had a few Aβ-positive vesicles. Retina 00-32 had a few large drusen and focal areas of free RPE cells in the subretinal space. Associated with these RPE abnormalities were a few Aβ-positive vesicles (Figure 4). This retina had minimal photoreceptor atrophy. Retina 00-1676 had many small and large drusen, and mild RPE atrophy (Figure 5A). While most of these drusen had no Aβ immunoreactivity (Figure 5B), a few had both Aβ-positive vesicles and smaller granular Aβ particles visible on confocal microscopy (Figure 5C).
None of the three exudative AMD retinas had Aβ-positive vesicles, but they also lacked large drusen. Retina 00-17 had a large exudative scar (Figure 6). In areas where RPE and photoreceptors were present (left side of image), the sub-RPE deposits were thin and Aβ negative. Similarly, none of the normal eyes, some of which had small drusen, had Aβ-positive vesicles (not shown).
The pathogenesis of AD and AMD may be related. Aβ, a major component of AD plaques, is also present in some drusen. Some of the Aβ in drusen is in vesicles and co-localizes with complement suggesting an association with inflammation . We sampled retina sections from 18 eyes, 9 with AMD and 9 normal, to determine the frequency and distribution of Aβ deposits in post mortem human eyes. We found Aβ-positive vesicles in drusen in 4 of 9 AMD eyes and none in normal eyes. The Aβ-positive vesicles were most numerous in GA eyes, near edges of atrophy. A few Aβ-positive vesicles were also present in drusen two AMD retinas with minimal RPE and photoreceptor atrophy.
The vesicles were detected with 3 different anti-Aβ antibodies. The label was blocked by pre-adsorption of each antibody with Aβ peptide, and was not present when Aβ antibody was omitted. These results strongly suggest that the vesicles contain Aβ.
The highest quantity of Aβ-containing vesicles occurred near edges of atrophy in GA eyes, the area at risk for expansion of GA [4,7,8]. The vesicles may either contribute to the pathogenesis of cell death in these RPE and photoreceptors at risk, or result from ongoing photoreceptor or RPE dysfunction or death. The vesicles are neither present in areas of severe atrophy, where RPE cells and photoreceptors are absent, nor in association with exudative scars lacking RPE cells and photoreceptors. These findings suggest that the vesicles may be cleared following RPE and photoreceptor apoptosis.
The most likely source of Aβ in drusen is the overlying RPE cells, which are known to deposit material into drusen. Like most other cell types, RPE cells express amyloid precursor protein  (APP), a membrane glycoprotein cleaved by proteases to produce the Aβ peptide. The biological functions of Aβ are unclear, but both Aβ multimers and fibrils can be neurotoxic . The quantitiy of Aβ present in drusen may depend upon alterations in protease activity producing Aβ and upon its clearance rate.
The finding of a few Aβ-positive vesicles associated with RPE abnormalities in early AMD suggests that Aβ may play a role in the early phases of AMD pathogenesis. However, as in the GA eyes, it is not clear whether the Aβ-positive vesicles are a cause or result of RPE and photoreceptor degeneration.
Many drusen in normal and AMD eyes lacked Aβ positive vesicles, so the vesicles are not a consistent component of drusen. Within AMD eyes, vesicles were apparent in both small (<63 μm) and large drusen.
Confocal imaging revealed that Aβ signal within drusen consisted not just of vesicles, but also of granular particles. It is possible that the granular particles assemble into vesicular structures, as Aβ peptides can self-assemble into spherical configurations .
All three Aβ antibodies labeled drusen from 4 of 9 AMD retinas. In contrast, these antibodies gave an inconsistent pattern of photoreceptor outer segment labeling. Future studies will employ additional Aβ antibodies and ELISA on retinal extracts to further investigate this issue.
Our data reveal that Aβ in drusen is associated with degenerating RPE and photoreceptors, suggesting that the Aβ may contribute to or result from degeneration. The finding of similarities in the pathogenesis of AMD and AD suggests that much can be learned about one disease from the other. Anti-Aβ therapies currently under development for AD may also prove useful for AMD. Thus, further investigation of the role of Aβ in AMD pathogenesis is warranted.
This work was supported by a Research to Prevent Blindness Career Development Award, International Retina Research Foundation, NIH (AG-10124, AG-11542, EY00417), Foundation Fighting Blindness, Pennsylvania Lions Foundation, F. M. Kirby Foundation, and the Paul and Evanina Bell Mackall Foundation Trust.
We thank W. Tang and J. Smith for their contributions to this work.
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